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Staphylococcus aureusis the main bacterial pathogen encountered in mediastinitis after cardiac surgical procedures; it remains a devastating complication with a high mortality rate. As neutrophils have a primordial role in the defense against staphylococcus infection and cardiopulmonary bypass (CPB) is known to induce immunosuppression, the aim of this study was to investigate CPB impact on neutrophil functions. Patients without known immunosuppression scheduled for cardiac surgery with CPB were included. Bone marrow and blood samples were harvested before, during, and after surgery. Neutrophil phenotypic maturation and functions (migration, adhesion, neutrophil extracellular trap [NET] release, reactive oxygen species (ROS) production, phagocytosis, and bacteria killing) were investigated. Two types of Staphylococcus aureusstrains (one from asymptomatic nasal carriage and another from mediastinitis infected tissues) were used to assess in vitro bacterial direct impact on neutrophils. We found that CPB induced a systemic inflammation with an increase in circulating mature neutrophils after surgery. Bone marrow sample analysis did not reveal any modification of neutrophil maturation during CPB. Neutrophil lifespan was significantly increased and functions such as NET release and ROS production were enhanced after CPB whereas bacteria killing and phagocytosis were not impacted. Results were similar with the two different isolates of Staphylococcus aureus. These data suggest that CPB induces a recruitment of mature neutrophils via a demargination process rather than impacting their maturation in the bone marrow. In addition, neutrophils are fully efficient after CPB and do not contribute to postoperative immunosuppression. Cardiopulmonary bypass does not immediately affect neutrophils functions.

•This population-based study of 245 patients with documented gonococcal infections in Western France in 2014–2016, found that:.•Almost 50% of cases are diagnosed by a general practitioner;.•The main clinical presentations include urethritis and pelvic inflammatory disease;.•Antibacterial treatment was appropriate in 100% of urethritis cases, and<50% of pelvic inflammatory diseases.

•The first study on antimicrobial susceptibility of 25 813 equine strains in France.•There was a high level of multidrug-resistant strains in equine clinical samples as reservoirs for human infection.•There is a need to promote more appropriate use of antibiotics by veterinarians.

The DaptoDP (NCT 2012-005699-33) study aimed to evaluate the pharmacokinetic parameters of daptomycin (DAP) in peritoneal dialysis-related peritonitis (PDRP) patients following intraperitoneal (IP) administration. The authors have already reported the findings on the 200-mg dosing and present here the follow-up results of the 300-mg dosing. The primary endpoint was a dialysate concentration of DAP above the effective concentration in situduring 6 hours of dwell time i.e., 16 mg/L. Secondary endpoints were to avoid the toxic threshold of 120 mg/L DAP and to be above 16 mg/L DAP for 2 hours in plasma. Pharmacokinetic parameters were evaluated on days 1 and 5. Safety data were evaluated on days 1 to 14 based on clinical and biological parameters. Daptomycin was administered in Nutrineal during 6 hours of dwell time for 14 days plus the usual antibiotic therapy in a separate dwell. Because the 200-mg dosing objectives were not reached, a higher DAP dose of 300 mg was tested in the next 3 patients. Effective dialysate and plasma concentrations were achieved at the 300-mg DAP dose with the plasma concentration well below the toxic threshold, even at steady state, during which the accumulation factor never exceeded 3. The optimal DAP dose of 300 mg daily by the IP route, as determined by the pharmacokinetic data, needs to be clinically confirmed prior to routine use. The peritoneal bioavailability of DAP supports using the IP route as an alternative to the intravenous route for peritonitis and systemic infections.

•100% and 98% of S. aureuswere susceptible to ceftaroline and ceftobiprole, respectively.•100% of CoNS were inhibited by ≤1μg/mL ceftaroline and 2μg/mL ceftobiprole, respectively.•MIC50/90values of ceftaroline were two- to four-fold lower than those of ceftobiprole.•At 4× MIC, marked bactericidal activity of ceftaroline and ceftobiprole against S. aureusand CoNS was observed.•More pronounced and rapid killing effect of ceftobiprole versus ceftaroline was observed against CoNS.

Background Antibiotics are preferentially delivered via the peritoneal route to treat peritoneal dialysis-related peritonitis (PDRP) to ensure that maximal concentrations are delivered to the site of infection. Our study focused on the pharmacokinetics of daptomycin (DAP) administered via the intraperitoneal (IP) route in patients with PDRP.Methods According to the DaptoDP protocol (Clinical Trial No. 2012-005699-33), IP DAP was administered daily, i.e., during the 6-h Nutrineal (Baxter Healthcare Corporation, Deerfield, IL, USA) dwell time period, for 14 days, in addition to administration of the antibiotics used for the usual care of patients with PDRP. The plasma and IP levels of DAP were measured on days 1 and 5. The tested dose was 200 mg/day. The principal endpoint was the dialysate concentration after 6 hours of dwell time > 16 mg/L (corresponding to 4 x minimum inhibitory concentration [MIC] for E. faecalis).Results Three participants were evaluated. On day 5, the IP concentrations after 6 hours of dwell time were between 6.3 and 23.4 mg/L, and the peak plasma concentrations were between 13.0 and 15.3 mg/L.Conclusion The results suggest that 200 mg/day is very likely sufficient for the treatment of PDRP by Staphylococcior Streptococciwhereas it could be insufficient to treat PRDP by Enterococci. The good peritoneal bioavailability of DAP was quantitatively established, suggesting that IP administration could also be used as an alternate route for patients with damaged venous access. No DAP accumulation that could lead to toxic concentrations after repeated administration is expected, even in anuric patients. The protocol will further continue to assess whether a higher dose achieves the pharmacokinetic objectives.

•88% of Enterobacteralesresistant to 3rdgeneration cephalosporins (3GC) remained susceptible to ceftolozane-tazobactam•Among ESBL producers, susceptibility to ceftolozane-tazobactam varies from 93% to only 13% for CTX-M and SHV-ESBL producers, respectively•In France, 95.6% of enterobacterial isolates responsible for infections (except urinary tract infections) collected prospectively were susceptible to ceftolozane-tazobactam•Etest® led to an underestimation of the ceftolozane-tazobactam MIC compared to a commercial broth microdilution method used as reference but the categorization agreement remained high (99%).

ABSTRACTIn Gram-negative bacteria, the active efflux is an important mechanism of antimicrobial resistance, but little is known about the Enterobacter cloacaecomplex (ECC). It is mediated primarily by pumps belonging to the RND (resistance-nodulation-cell division) family, and only AcrB, part of the AcrAB-TolC tripartite system, was characterized in ECC. However, detailed genome sequence analysis of the strain E. cloacaesubsp. cloacaeATCC 13047 revealed to us that 10 other genes putatively coded for RND-type transporters. We then characterized the role of all of these candidates by construction of corresponding deletion mutants, which were tested for their antimicrobial susceptibility to 36 compounds, their virulence in the invertebrate Galleria mellonellamodel of infection, and their ability to form biofilm. Only the ΔacrBmutant displayed significantly different phenotypes compared to that of the wild-type strain: 4- to 32-fold decrease of MICs of several antibiotics, antiseptics, and dyes, increased production of biofilm, and attenuated virulence in G. mellonella. In order to identify specific substrates of each pump, we individually expressed in transall operons containing an RND pump-encoding gene into the ΔacrBhypersusceptible strain. We showed that three other RND-type efflux systems (ECL_00053-00055, ECL_01758-01759, and ECL_02124-02125) were able to partially restore the wild-type phenotype and to superadd to and even enlarge the broad range of antimicrobial resistance. This is the first global study assessing the role of all RND efflux pumps chromosomally encoded by the ECC, which confirms the major role of AcrB in both pathogenicity and resistance and the potential involvement of other RND-type members in acquired resistance.

ABSTRACTAn interregional surveillance program was conducted in the northwestern part of France to determine the prevalence of carbapenem-nonsusceptible Enterobacteriaceae(CNSE) isolates and their susceptibility to ceftazidime-avibactam and aztreonam-avibactam combinations. Nonduplicate CNSE clinical isolates were prospectively collected from six hospitals between June 2012 and November 2013. MICs of ceftazidime and aztreonam, alone or combined with a fixed concentration of avibactam (4 μg/ml), and those of carbapenems (comparator agents) were determined. MICs of ertapenem in combination with phenylalanine arginine-naphthylamide dihydrochloride (PAβN) were also determined to assess active efflux. Genes encoding carbapenemases, plasmid-mediated AmpC enzymes, extended-spectrum β-lactamases (ESBLs), and major outer membrane proteins (OMPs) were amplified and sequenced. OMPs were also extracted for SDS-PAGE analysis. Among the 139 CNSE isolates, mainly Enterobacterspp. and Klebsiella pneumoniae, 123 (88.4%) were ertapenem nonsusceptible, 12 (8.6%) exhibited reduced susceptibility to all carbapenems, and 4 Proteeaeisolates (2.9%) were resistant to imipenem. Carbapenemase production was detected in only two isolates (producing OXA-48 and IMI-3). In contrast, OMP deficiency, in association with AmpCs and/or ESBLs (mainly CTX-M-9, SHV-12, and CTX-M-15), was largely identified among CNSE isolates. The ceftazidime-avibactam and aztreonam-avibactam combinations exhibited potent activity against CNSE isolates (MIC50/MIC90, 1/1 μg/ml and 0.5/0.5 μg/ml, respectively) compared to that of ceftazidime and aztreonam alone (MIC50/MIC90, 512/512 μg/ml and 128/512 μg/ml, respectively). This study reveals the in vitroactivity of ceftazidime-avibactam and aztreonam-avibactam combinations against a large collection of porin-deficient enterobacterial isolates that are representative of the CNSE recovered in the northern part of France.

ABSTRACTEnterobacter cloacaecomplex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist β-lactams mainly by producing the AmpC β-lactamase enzyme. AmpC expression is highly inducible in the presence of some β-lactams, but the underlying genetic regulation, which is intricately linked to peptidoglycan recycling, is still poorly understood. In this study, we constructed different mutant strains that were affected in genes encoding enzymes suspected to be involved in this pathway. As expected, the inactivation of ampC, ampR(which encodes the regulator protein of ampC), and ampG(encoding a permease) abolished β-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression of ampCin different ways: one involving NagZ (a N-acetyl-β-d-glucosaminidase) and another independent of NagZ. Unlike the model established for Pseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutive ampCoverexpression in ECC, whereas it caused AmpC-mediated high-level β-lactam resistance, suggesting a post-transcriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of a dacBdeletion mutant confirmed these results. Lastly, analysis of 37 ECC clinical isolates showed that amino acid changes in the AmpD sequence were likely the most crucial event involved in the development of high-level β-lactam resistance in vivoas opposed to P. aeruginosawhere dacBmutations have been commonly found. These findings bring new elements for a better understanding of β-lactam resistance in ECC, which is essential for the identification of novel potential drug targets.

ABSTRACTTigecycline (TIG) is approved for use for the treatment of complicated intra-abdominal infections, skin and skin structure infections, as well as pneumonia. Acquired resistance or reduced susceptibility to TIG has been observed in Gram-negative rods, has seldom been reported in Gram-positive organisms, and has not yet been reported in Enterococcus faecium. Using the serial passage method, in vitromutant AusTig and in vitromutants HMtig1 and HMtig2 with decreased TIG susceptibility (MICs, 0.25 μg/ml) were obtained from strains E. faeciumAus0004 and HM1070 (MICs, 0.03 μg/ml), respectively. In addition, two vancomycin-resistant E. faeciumclinical isolates (EF16 and EF22) with reduced susceptibility to TIG (MICs, 0.5 and 0.25 μg/ml, respectively) were studied. Compared to the wild-type strains, the in vitromutants also showed an increase in the MICs of other tetracyclines. An efflux mechanism did not seem to be involved in the reduced TIG susceptibility, since the presence of efflux pump inhibitors (reserpine or pantoprazole) did not affect the MICs of TIG. Whole-genome sequencing of AusTig was carried out, and genomic comparison with the Aus0004 genome was performed. Four modifications leading to an amino acid substitution were found. These mutations affected the rpsJgene (efau004_00094, coding for the S10 protein of the 30S ribosomal subunit), efau004_01228(encoding a cation transporter), efau004_01636(coding for a hypothetical protein), and efau004_02455(encoding the l-lactate oxidase). The four other strains exhibiting reduced TIG susceptibility were screened for the candidate mutations. This analysis revealed that three of them showed an amino acid substitution in the same region of the RpsJ protein. In this study, we characterized for the first time genetic determinants linked to reduced TIG susceptibility in enterococci.

ABSTRACTCritically ill patients who require renal replacement therapy (RRT) are vulnerable to catheter-related bloodstream infections (CRBSI). This study compared the risks of dialysis catheter infection according to the choice of locking solution in the intensive care unit (ICU). A prospective quasi-experimental study with marginal structural models (MSM) and 2:1 greedy propensity-score matching (PSM) was conducted at nine university-affiliated hospitals and three general hospitals. A total of 596 critically ill patients received either saline solution or heparin lock solution (the standard of care [SOC]) from 2004 to 2007 in the Cathedia cohort (n= 464 for MSM; n= 124 for PSM) or 46.7% citrate lock from 2011 to 2012 in the citrate (CLock) cohort (n= 132 for MSM; n= 62 for PSM) to perform RRT using intermittent hemodialysis. Catheter-tip colonization and CRBSI were analyzed. The mean duration (standard deviation [SD]) of catheterization was 7.1 days (6.1) in the SOC group and 7.0 days (5.9) in the CLock group (P= 0.84). The risk of dialysis catheter-tip colonization was lower in the CLock group (20.5 versus 38.7 per 1,000 catheter-days in the SOC group; hazard ratio [HR] from MSM, 0.73; 95% confidence interval [CI], 0.57 to 0.93; P< 0.02). Consistent findings were found from PSM (HR, 0.46; 95% CI, 0.22 to 0.95; P< 0.04). The risk of CRBSI was nonsignificantly different in the CLock group (1.1 versus 1.8 per 1,000 catheter-days in the SOC group; HR from MSM, 0.48; 95% CI, 0.12 to 1.87; P= 0.29). By reducing the risk of catheter-tip colonization, citrate lock has the potential to improve hemodialysis safety in the ICU. Additional studies are warranted before the routine use of citrate locks can be recommended in the ICU.

ABSTRACTRhodococcus equi, the causal agent of rhodococcosis, is a major pathogen of foals and is also responsible for severe infections in immunocompromised humans. Of great concern, strains resistant to currently used antibiotics have emerged. As the number of drugs that are efficient in vivois limited because of the intracellular localization of the bacterium inside macrophages, new active but cell-permeant drugs will be needed in the near future. In the present study, we evaluated, by in vitroand ex vivoexperiments, the ability of the alpha-helical equine antimicrobial peptide eCATH1 to kill intracellular bacterial cells. Moreover, the therapeutic potential of the peptide was assessed in experimental rhodococcosis induced in mice, while the in vivotoxicity was evaluated by behavioral and histopathological analysis. The study revealed that eCATH1 significantly reduced the number of bacteria inside macrophages. Furthermore, the bactericidal potential of the peptide was maintained in vivoat doses that appeared to have no visible deleterious effects for the mice even after 7 days of treatment. Indeed, daily subcutaneous injections of 1 mg/kg body weight of eCATH1 led to a significant reduction of the bacterial load in organs comparable to that obtained after treatment with 10 mg/kg body weight of rifampin. Interestingly, the combination of the peptide with rifampin showed a synergistic interaction in both ex vivoand in vivoexperiments. These results emphasize the therapeutic potential that eCATH1 represents in the treatment of rhodococcosis.

ABSTRACTAs opposed to Enterococcus faecalis, which is intrinsically resistant to lincosamides, streptogramins A, and pleuromutilins (LSAP phenotype) by production of the ABC protein Lsa(A), Enterococcus faeciumis naturally susceptible. Since this phenotype may be selected for in vivoby quinupristin-dalfopristin (Q-D), the aim of this study was to investigate the molecular mechanism of acquired LSAP resistance in E. faecium. Six LSAP-resistant in vitromutants of E. faeciumHM1070 as well as three different pairs of clinical isolates (pre- and postexposure to Q-D) were studied. The full genome sequence of an in vitromutant (E. faeciumUCN90B) was determined by using 454 sequencing technology and was compared with that of the parental strain. Single-nucleotide replacement was carried out to confirm the role of this mutation. By comparative genomic analysis, a point mutation was found within a 1,503-bp gene coding for an ABC homologue showing 66% amino acid identity with Lsa(A). This mutation (C1349T) led to an amino acid substitution (Thr450Ile). An identical mutation was identified in all in vitroand in vivoresistant strains but was not present in susceptible strains. The wild-type allele was named eat(A) (for EnterococcusABC transporter), and its mutated allelic variant was named eat(A)v. The introduction of eat(A)vfrom UCN90B into HM1070 conferred the LSAP phenotype, whereas that of eat(A) from HM1070 into UCN90B restored susceptibility entirely. This is the first description of the molecular mechanism of acquired LSAP resistance in E. faecium. Characterization of the biochemical mechanism of resistance and the physiological role of this ABC protein need further investigations.

ABSTRACTEnterococcus faeciumUCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanNwas obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to the d-alanine:d-serine VanL ligase. The organization of the vanNgene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of the vanCoperons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending in d-serine and d,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation to E. faecium. This is the first report of transferable d-Ala-d-Ser-type resistance in E. faecium.

ABSTRACTStreptococcus agalactiaeUCN70, isolated from a vaginal swab obtained in New Zealand, is resistant to lincosamides and streptogramins A (LSAphenotype) and also to tiamulin (a pleuromutilin). By whole-genome sequencing, we identified a 5,224-bp chromosomal extra-element that comprised a 1,479-bp open reading frame coding for an ABC protein (492 amino acids) 45% identical to Lsa(A), a protein related to intrinsic LSAresistance in Enterococcus faecalis. Expression of this novel gene, named lsa(C), in S. agalactiaeBM132 after cloning led to an increase in MICs of lincomycin (0.06 to 4 μg/ml), clindamycin (0.03 to 2 μg/ml), dalfopristin (2 to >32 μg/ml), and tiamulin (0.12 to 32 μg/ml), whereas no change in MICs of erythromycin (0.06 μg/ml), azithromycin (0.03 μg/ml), spiramycin (0.25 μg/ml), telithromycin (0.03 μg/ml), and quinupristin (8 μg/ml) was observed. The phenotype was renamed the LSAP phenotype on the basis of cross-resistance to lincosamides, streptogramins A, and pleuromutilins. This gene was also identified in similar genetic environments in 17 other S. agalactiaeclinical isolates from New Zealand exhibiting the same LSAP phenotype, whereas it was absent in susceptible S. agalactiaestrains. Interestingly, this extra-element was bracketed by a 7-bp duplication of a target site (ATTAGAA), suggesting that this structure was likely a mobile genetic element. In conclusion, we identified a novel gene, lsa(C), responsible for the acquired LSAP resistance phenotype in S. agalactiae. Dissection of the biochemical basis of resistance, as well as demonstration of in vitromobilization of lsa(C), remains to be performed.

Enterococcus faeciuma développé une multirésistance aux antibiotiques dont les glycopeptides. Les glycopeptides (vancomycine et teicoplanine) agissent sur les entérocoques en se fixant sur les précurseurs de la paroi bactérienne dont ils empêchent ainsi la formation. La résistance est due à l’acquisition d’un opéron de gènes coopérant pour synthétiser des précurseurs sans affinité pour les glycopeptides. Des épidémies sont survenues récemment en milieu hospitalier dues à des souches de E. faeciumappartenant à un complexe clonal (CC17) adapté à l’hôpital. Les épidémies peuvent être contrôlées par des mesures appropriées et de nouveaux antibiotiques qui sont disponibles. La dissémination de souches adaptées aux conditions hospitalières nécessite leur surveillance étroite.

Resistance to quinolones and fluoroquinolones has been increasingly reported among human and veterinary isolates during the last three decades related to their wide clinical use. Until recently, the mechanisms of resistance to quinolones in Enterobacteriaceae were believed to be only chromosome-encoded, i.e. related to modifications of the molecular targets (DNA gyrase and topoisomerase IV), decreased outer-membrane permeability (porin defect) and overexpression of naturally-occurring efflux. However, emergence of plasmid-mediated quinolone resistance (PMQR) has been reported since 1998. Three mechanisms are known to date: Qnr proteins, aminoglycoside acetyltransferase AAC(6)-Ib-cr, and efflux pump QepA. The Qnr proteins protect DNA gyrase and type IV topoisomerase from quinolone inhibition. Four types of Qnr protiens have been reported: QnrA (six variants), QnrB (19 variants), QnrC (one variant), and QnrS (three variants). The AAC(6)-Ib-cr determinant acetylates several fluoroquinolones, such as norfloxacin and ciprofloxacin. The protein AAC(6)-Ib-cr contains two amino acid substitutions as compared to the wild-type enzyme AAC(6)-Ib. Both Qnr and AAC(6)-Ib proteins have been reported worldwide. Lately reported, the plasmid-encoded QepA efflux pump may extrude hydrophilic fluoroquinolones (eg. norfloxacin, ciprofloxacin, and enrofloxacin).

ABSTRACTOne hundred twenty-one extended-spectrum β-lactamse-producing enterobacterial clinical isolates were screened for the qepAgene. A single CTX-M-15-positive Escherichia coliisolate (0.8%) that produced the putative pump QepA2 was identified. This qepA2gene was located onto a 90-kb mobilizable plasmid that conferred reduced susceptibility to hydrophilic fluoroquinolones.

ABSTRACTA novel QnrB-like plasmid-mediated resistance determinant, QnrB19, was identified from an Escherichia coliclinical isolate from Colombia. Its gene was associated with an ISEcp1-like insertion element that did not act as a promoter for its expression. Using an in vitro model of transposition, we showed that the ISEcp1-like element was able to mobilize the qnrB19gene.

Carbapenem resistance results mostly from the expression of acquired carbapenem-hydrolyzing oxacillinases in Acinetobacter baumannii. The blaOXA-23oxacillinase gene is increasingly reported worldwide and may represent an emerging threat. Our goal was to identify the progenitor of that carbapenemase gene. A collection of 50 Acinetobacter sp. strains corresponding to several Acinetobacter species was screened for blaOXA-23-like genes by PCR and hybridization techniques. Five Acinetobacter radioresistens isolates that were susceptible to carbapenems harbored chromosomally encoded blaOXA-23-like genes. A similar plasmid backbone was identified in several blaOXA-23-positive A. baumannii and A. radioresistens isolates, further strengthening the vectors of exchanges for these blaOXA-23-like genes. Therefore, A. radioresistens, a commensal bacterial species which is identified on the skin of hospitalized and healthy patients (a property shared with A. baumannii), was identified as the source of the blaOXA-23gene.

ABSTRACTCarbapenem resistance results mostly from the expression of acquired carbapenem-hydrolyzing oxacillinases in Acinetobacter baumannii. The blaOXA-23oxacillinase gene is increasingly reported worldwide and may represent an emerging threat. Our goal was to identify the progenitor of that carbapenemase gene. A collection of 50 Acinetobactersp. strains corresponding to several Acinetobacterspecies was screened for blaOXA-23-like genes by PCR and hybridization techniques. Five Acinetobacter radioresistensisolates that were susceptible to carbapenems harbored chromosomally encoded blaOXA-23-like genes. A similar plasmid backbone was identified in several blaOXA-23-positive A. baumanniiand A. radioresistensisolates, further strengthening the vectors of exchanges for these blaOXA-23-like genes. Therefore, A. radioresistens, a commensal bacterial species which is identified on the skin of hospitalized and healthy patients (a property shared with A. baumannii), was identified as the source of the blaOXA-23gene.

ABSTRACTBy studying the β-lactamase content of several Acinetobacterspp. isolates from Argentina, producing the expanded-spectrum β-lactamases (ESBL) VEB-1a or PER-2, a novel Ambler class A β-lactamase gene was identified. It encoded the narrow-spectrum β-lactamase SCO-1, whose activity was inhibited by clavulanic acid. SCO-1 hydrolyzes penicillins at a high level and cephalosporins and carbapenems at a very low level. β-Lactamase SCO-1 was identified from unrelated VEB-1a-positive or PER-2-positive Acinetobacterspp. isolates recovered from three hospitals. The blaSCO-1gene was apparently located on a plasmid of ca. 150 kb from all cases but was not associated with any ESBL-encoding gene. The G+C content of the blaSCOgene was 52%, a value that does not correspond to that of the A. baumanniigenome (39%). β-Lactamase SCO-1 shares 47% amino acid identity with CARB-5 and ca. 40% with the enzymes TEM, SHV, and CTX-M. A gene encoding a putative resolvase was identified downstream of the blaSCO-1gene, but its precise way of acquisition remains to be determined.

The plasmid-mediated quinolone resistance determinant QnrS1 was identified in non-clonally related Enterobacter cloacae isolates in association with a transferable narrow-spectrum {szligbeta}-lactam resistance marker. Cloning experiments allowed the identification of a novel Ambler class A {szligbeta}-lactamase, named LAP-1. It shares 62 and 61% amino acid identity with the most closely related {szligbeta}-lactamases, TEM-1 and SHV-1, respectively. It has a narrow-spectrum hydrolysis of {szligbeta}-lactams and is strongly inhibited by clavulanic acid and sulbactam and, to a lesser extent, by tazobactam. Association of the blaLAP-1gene with the qnrS1 gene was identified in E. cloacae isolates from France and Vietnam. These genes were plasmid located and associated with similar insertion sequences but were not associated with sul1-type class 1 integrons, as opposed to the qnrA genes.

ABSTRACTThe plasmid-mediated quinolone resistance determinant QnrS1 was identified in non-clonally related Enterobacter cloacaeisolates in association with a transferable narrow-spectrum β-lactam resistance marker. Cloning experiments allowed the identification of a novel Ambler class A β-lactamase, named LAP-1. It shares 62 and 61% amino acid identity with the most closely related β-lactamases, TEM-1 and SHV-1, respectively. It has a narrow-spectrum hydrolysis of β-lactams and is strongly inhibited by clavulanic acid and sulbactam and, to a lesser extent, by tazobactam. Association of the blaLAP-1gene with the qnrS1gene was identified in E. cloacaeisolates from France and Vietnam. These genes were plasmid located and associated with similar insertion sequences but were not associated with sul1-type class 1 integrons, as opposed to the qnrAgenes.

ABSTRACTThe species belonging to the Acinetobactergenus are currently reported as opportunistic pathogens in hospitalized patients with underlying predispositions. However, except for the Acinetobacter calcoaceticus-Acinetobacter baumanniicomplex, the identification of other species is frequently unreliable, especially for Acinetobacter ursingiiand Acinetobacter schindleri, newly described in 2001. Thus, the clinical significance, phenotypic features, and antimicrobial susceptibilities of these two misidentified species remain unclear. Of 456 Acinetobactersp. clinical strains isolated from 2002 to 2005 in Henri Mondor Hospital, 15 isolates (10 A. ursingiiand 5 A. schindleriisolates) were studied. They were characterized using a phenotypic approach (API 20 NE and VITEK 2 systems), 16S rRNA gene sequencing, and susceptibility to antimicrobial agents with evaluation of impact in clinical relevance. The two corresponding type strains were also included for comparison. All isolates were identified to the species level using molecular tools, whereas the phenotypic methods remained unreliable due to the absence of these two species in the manufacturers' databases. However, the API 20 NE system appeared to be a reasonably reliable phenotypic alternative for the identification of A. ursingiiwhen the numerical code 0000071 was found. Conversely, no discriminative phenotypic alternative existed for A. schindleriisolates. Concerning antimicrobial susceptibility, A. ursingiistrains appeared to be more resistant to antibiotics than A. schindleristrains, which could imply therapeutic consequences. Finally, the prevalence of infections caused by A. ursingiiand A. schindleri(representing 9.7% and 4.8% of non-A. calcoaceticus-A. baumanniicomplex strains, respectively) seems to be underestimated.

ABSTRACTFrom 2008 to 2013, 39 Helcococcus kunziistrains were collected from human clinical specimens (79% from foot ulcers), and 85% of the 39 patients were infected. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry and molecular methods accurately identified all isolates. This large study of clinical observations confirms the potential pathogenic role of H. kunzii.

Objective To describe the characteristics, management and outcomes of hospitalised patients with Clostridioides difficileinfection (CDI) treated with and without fidaxomicin.Methods This prospective, multicentre, observational study (DAFNE) enrolled hospitalised patients with CDI, including 294 patients treated with fidaxomicin (outcomes recorded over a 3-month period) and 150 patients treated with other CDI therapies during three 1-month periods. The primary endpoint was baseline and CDI characteristics of fidaxomicin-treated patients.Results At baseline, the fidaxomicin-treated population included immunocompromised patients (39.1%) and patients with severe (59.2%) and recurrent (36.4%) CDI. Fidaxomicin was associated with a high rate of clinical cure (92.2%) and low CDI recurrence (16.3% within 3 months). Clinical cure rates were ≥90% in patients aged ≥65 years, those receiving concomitant antibiotics and those with prior or severe CDI. There were 121/296 (40.9%) patients with adverse events (AEs), 5.4% with fidaxomicin-related AEs and 1.0% with serious fidaxomicin-related AEs. No fidaxomicin-related deaths were reported.Conclusions Fidaxomicin is an effective and well-tolerated CDI treatment in a real-world setting in France, which included patients at high risk of adverse outcomes. Trial registration: Description of the use of fidaxomicin in hospitalised patients with documented Clostridium difficileinfection and the management of these patients (DAFNE), NCT02214771, www.ClinicalTrials.gov.

•Dalbavancin is effective for PJI•Dalbavancin is safe•Optimal dosage should be defined

Enterococcus faeciumhas become a major opportunistic pathogen with the emergence of vancomycin-resistant enterococci (VRE). As part of the gut microbiota, they have to cope with numerous stresses, including effects of antibiotics and other xenobiotics, especially in patients hospitalized in intensive care units (ICUs) who receive many medications. The aim of this study was to investigate the impact of the most frequently prescribed xenobiotics for ICU patients on fitness, pathogenicity, and antimicrobial resistance of the vanB-positive E. faeciumAus0004 reference strain.

Genome changes are central to the adaptation of bacteria, especially under antibiotic pressure. The aim of this study was to report phenotypic and genomic adaptations undergone by an Enterobacter hormaecheiclinical strain that became highly resistant to key antimicrobials during a 4-month period in a patient hospitalized in an intensive care unit (ICU). All six clinical E. hormaecheistrains isolated in one ICU-hospitalized patient have been studied. MICs regarding 17 antimicrobial molecules have been measured.

The clinical benefit of carbapenems against carbapenemase-producing Enterobacteriaceae(CPE) remains in question. MICs of imipenem (IMP) and ertapenem (ERT) against isogenic derivatives of the wild-type strain Escherichia coliCFT073 producing KPC-3, OXA-48, or NDM-1 were 0.25, 2, 16, and 64 mg/liter for IMP and 0.008, 0.5, 8, and 64 mg/liter for ERT, respectively. Swiss ICR-strain mice with peritonitis were treated for 24 h with IMP or ERT. Despite a limited duration of time during which free antibiotic concentrations were above the MIC (down to 0% for the NDM-1-producing strain), IMP and ERT significantly reduced bacterial counts in spleen and peritoneal fluid at 24 h (P< 0.

Major facilitator superfamily (MFS) efflux pumps have been shown to be important for bacterial cells to cope with biocides such as chlorhexidine (CHX), a widely used molecule in hospital settings. In this work, we evaluated the role of two genes, smvAand smvR, in CHX resistance in Enterobacter cloacaecomplex (ECC). smvAencodes an MFS pump whereas smvR, located upstream of smvA,codes for a TetR-type transcriptional repressor.

Fosfomycin tromethamine activity is well established for oral treatment of uncomplicated lower urinary tract infections, but little is known about its potential efficacy in pyelonephritis. Ascending pyelonephritis was induced in mice infected with 6 strains of Escherichia coli(fosfomycin MICs, 1 μg/ml to 256 μg/ml). The urine pH was 4.5 before infection and 5.5 to 6.0 during infection.

The study evaluated the in vitroactivity of ceftolozane-tazobactam (C/T) against 94 unique clinical isolates of Enterobacter cloacaecomplex (ECC). No difference was observed according to the ECC cluster.

ABSTRACTQuaternary amine compounds (QAC) are potent antimicrobials used to prevent the spread of pathogenic bacteria. While they are known for their membrane-damaging properties, QAC action has been suggested to extend beyond the surface to intracellular targets. Here we characterize the range of action of the QAC biocide benzalkonium chloride (BZK) against the bacterial pathogen Acinetobacter baumannii. At high concentrations, BZK acts through membrane disruption, but at low concentrations we show that wide-spread protein aggregation is associated with BZK-induced cell death. Resistance to BZK is found to develop through ribosomal protein mutations that protect A. baumanniiagainst BZK-induced protein aggregation. The multifunctional impact of BZK led us to discover that alternative QAC structures, with low human toxicity, retain potent action against multidrug-resistant A. baumannii, Staphylococcus aureus, and Clostridium difficileand present opportunities for their development as antibiotics.IMPORTANCEQuaternary amine compounds (QACs) are widely used to prevent the spread of bacterial pathogens, but our understanding of their mode of action is incomplete. Here we describe disruption of bacterial proteostasis as an unrecognized action of QAC antimicrobial action and uncover the potential of diverse QAC structures to act as multitarget antibiotics.

ABSTRACTThe extensive use of antibiotics has resulted in a situation where multidrug-resistant pathogens have become a severe menace to human health worldwide. A deeper understanding of the principles used by pathogens to adapt to, respond to, and resist antibiotics would pave the road to the discovery of drugs with novel mechanisms. For bacteria, antibiotics represent clinically relevant stresses that induce protective responses. The recent implication of regulatory RNAs (small RNAs [sRNAs]) in antibiotic response and resistance in several bacterial pathogens suggests that they should be considered innovative drug targets. This minireview discusses sRNA-mediated mechanisms exploited by bacterial pathogens to fight against antibiotics. A critical discussion of the newest findings in the field is provided, with emphasis on the implication of sRNAs in major mechanisms leading to antibiotic resistance, including drug uptake, active drug efflux, drug target modifications, biofilms, cell walls, and lipopolysaccharide (LPS) biosynthesis. Of interest is the lack of knowledge about sRNAs implicated in Gram-positive compared to Gram-negative bacterial resistance.

ABSTRACTEnterococcus faeciumhas emerged as a major opportunistic pathogen for 2 decades with the spread of hospital-adapted multidrug-resistant clones. As members of the intestinal microbiota, they are subjected to numerous bacterial stresses, including antibiotics at subinhibitory concentrations (SICs). Since fluoroquinolones are extensively prescribed, SICs are very likely to occur in vivo, with potential effects on bacterial metabolism with subsequent modulation of opportunistic traits. The aim of this study was to evaluate globally the impact of SICs of ciprofloxacin on antimicrobial resistance and pathogenicity of E. faecium. Transcriptomic analysis was performed by RNA sequencing (RNA-seq) (HiSeq 2500; Illumina) using the vanB-positive reference strain E. faeciumAus0004 in the absence or presence of ciprofloxacin SIC (0.38 mg/liter, i.e., 1/8 of the MIC). Several genetic and phenotypic tests were used for validation. In the presence of ciprofloxacin SIC, 196 genes were significantly induced, whereas 286 genes were significantly repressed, meaning that 16.8% of the E. faeciumgenome was altered. Among upregulated genes, EFAU004_02294 (fold change, 14.3) encoded a protein (Qnr of E. faecium[EfmQnr]) homologue of Qnr proteins involved in quinolone resistance in Gram-negative bacilli. Its implication in intrinsic and adaptive fluoroquinolone (FQ) resistance in E. faeciumwas experimentally ascertained. Moreover, EFAU004_02292, coding for the collagen adhesin Acm, was also induced by the SIC of ciprofloxacin (fold change, 8.2), and higher adhesion capabilities were demonstrated phenotypically. Both EfmQnr and Acm determinants may play an important role in the transition from a commensal to a pathogenic state of E. faeciumthat resides in the gut of patients receiving fluoroquinolone therapy.

The quest to stem antibiotic resistance might be exacerbated by enhanced fitness and virulence displayed by the drug-resistant microbes.

&lt;it>Objectives&lt;/it>: To investigate quinolone resistance mechanisms in an &lt;it>Escherichia coli&lt;/it> clinical isolate (Ar2) resistant to ofloxacin but susceptible to nalidixic acid selected after 10 days of ofloxacin therapy in a patient with prostatitis. &lt;it>Methods&lt;/it>: Molecular typing (ERIC-PCR and RAPD), antibiotic susceptibility and &lt;it>gyrA&lt;/it>, &lt;it>gyrB&lt;/it>, &lt;it>parC&lt;/it> and &lt;it>parE&lt;/it> QRDR sequences were compared for &lt;it>E. coli&lt;/it> Ar2 and a wild-type &lt;it>E. coli&lt;/it> (Ar1) isolated 2 months earlier in the same patient. Ofloxacin-resistant mutants were selected &lt;it>in vitro&lt;/it> in order to reproduce the mutations observed and the original phenotype. &lt;it>Results&lt;/it>: The two strains were similar with regard to antibiotic susceptibility except quinolones and for ERIC-PCR and RAPD patterns, suggesting a clonal relationship and acquisition of quinolone resistance by chromosomal mutation. Quinolone MICs were 3, 0.12, 0.05 and 0.02 mg/L of nalidixic acid, ofloxacin, levofloxacin and ciprofloxacin, respectively, for &lt;it>E. coli&lt;/it> Ar1 and 6, 32, 8 and 1 mg/L, respectively, for &lt;it>E. coli&lt;/it> Ar2. The strain Ar2 harboured two substitutions, Gly-81→Asp in GyrA and Ser-80→Arg in ParC. Introduction into &lt;it>E. coli&lt;/it> Ar2 of the wild-type &lt;it>gyrA&lt;/it> fully complemented fluoroquinolone resistance. Although the strain was not a hypermutator, ofloxacin first-step resistant mutants with &lt;it>gyrA&lt;/it> mutations were easily obtained from &lt;it>E. coli&lt;/it> Ar1 and 25% of them were at codon 81. &lt;it>In vitro&lt;/it> stepwise combination of Gly-81→Asp in GyrA and Ser-80→Arg in ParC reproduced the original phenotype in &lt;it>E. coli&lt;/it> KL16. &lt;it>Conclusions&lt;/it>: A double topoisomerase mutant was selected &lt;it>in vivo&lt;/it> by 10 days ofloxacin. The mutations were originally combined for a result of ofloxacin resistance but nalidixic acid susceptibility.